Rejection Grade Research Articles

Implication of long non-coding RNAs in the pathophysiology of cardiac rejection and its accuracy in diagnosis

Abstract Background The complementation of the endomyocardial biopsy or even its replacement by the use of a non-invasive method is one of the great challenges in the investigation of cardiac rejection. However, the limitations of the serum markers identified so far limit their use in clinical practice. In recent years, many long non-coding RNAs (lncRNAs) have been identified along with their role in different biological functions. However, the utility of lncRNAs in cardiac rejection has not been explored. Purpose We aimed to determine the expression of lncRNAs in serum of patient with cardiac rejection and evaluate the diagnostic capacity as potential biomarkers for the non-invasive detection of mild (grade 1R) or moderate/severe (grade ≥2R) cellular rejection. Methods RNA sequencing analysis (Illumina HiSeq 2500) was performed on forty serum samples from transplant recipients undergoing routine endomyocardial biopsies. Twenty eight samples were diagnosed with RCA (RCA Grade 1R, n=16; and RCA Grade ≥2R, n=12) and 12 samples had no diagnosed rejection. Results Of the 10,144 lncRNAs detected in serum, 5.1% presented differential expression in patients with cardiac rejection of Grade ≥2R. Fifty-six molecules showed excellent diagnostic ability in moderate-severe rejection (AUC >0.900) and fifty-one molecule were even able to detect mild rejection with good diagnostic ability for moderate-severe rejection (AUC >0.800). Three lncRNAs stand out: EGFR-AS1 (≥2R, AUC=0.871, p<0.01; 1R, AUC=0.921, p<0.0001), AC008649.2 (≥2R, AUC=0.867, p<0.01; 1R, AUC=0.807, p<0.01) and AP001652.1 (≥2R, AUC=0.848, p<0.01; 1R, AUC=0.855, p<0.01). Conclusions A large number of lncRNAs with excellent diagnostic capacity for acute cellular rejection have been identified. In addition, we highlight 3 long non-coding (EGFR-AS1, AC008649.2 and AP001652.1) that maintain a good diagnostic capacity even in the mildest degrees. Therefore, we propose these non-coding RNAs as potential biomarkers of cardiac rejection. Although the function of these non-coding RNAs in acute cellular rejection is unknown, it is important to take into account in future studies the pathways in which they participate in order to elucidate the mechanisms involved in the development of this pathology. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Institute of Health Carlos III European Regional Development Fund (ERDF)

Combining serum miR-144-3p and miR-652-3p as potential biomarkers for the early diagnosis and stratification of acute cellular rejection in heart transplantation patients

Abstract Background There is a dire need for specific, noninvasive biomarkers that can accurately and early detect cardiac acute cellular rejection (ACR). Previously, we described miR-144-3p as an excellent candidate to detect Grade ≥2R ACR. Purpose Now, we investigated the combination of miR-144-3p with other differentially expressed serum miRNA that we previously described to improve diagnostic accuracy, mainly in mild rejection to avoid reaching severe stages. Methods We selected miR-652-3p, miR-6747-3p and miR-221-3p from a preliminary RNA-seq study to be validated by RT-qPCR on 212 consecutive serum samples from transplantation recipients undergoing routine endomyocardial biopsies to subsequently combine them with miR-144-3p results and investigate their diagnostic capability. Results We confirmed the miR-652-3p overexpression (p<0.0001) and its capability to discriminate between patients with and without ACR of any grade (p<0.0001), but not miR-6747-3p or miR-221-3p. The combined serum levels of miR-144-3p and miR-652-3p were significantly higher in patients with rejection regardless of posttransplantation time (p<0.0001). This combination resulted in a diagnostic efficacy for 1R (AUC=0.794) and ≥2R (AUC=0.892) (p<0.0001) that was superior to each biomarker alone. Furthermore, it was a strong independent predictor of ACR for 1R (odds ratio of 10.950, p<0.0001) and ≥2R (odds ratio of 14.289, p<0.01). Conclusions We demonstrated that an appropriate combination of blood-based biomarkers can exhibit greater efficiency for cardiac rejection diagnosis. The combined detection of abnormal expression of miR-144-3p and miR-652-3p in the serum of ACR patients can improve the diagnostic sensitivity of rejection at the early stage and contribute to increasing the diagnostic accuracy, mainly in the lower rejection grades. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Institute of Health Carlos III European Regional Development Fund (ERDF)

Improving the ischemia-reperfusion injury in vascularized composite allotransplantation: Clinical experience and experimental implications.

Long-time ischemia worsening transplant outcomes in vascularized composite allotransplantation (VCA) is often neglected. Ischemia-reperfusion injury (IRI) is an inevitable event that follows reperfusion after a period of cold static storage. The pathophysiological mechanism activates local inflammation, which is a barrier to allograft long-term immune tolerance. The previous publications have not clearly described the relationship between the tissue damage and ischemia time, nor the rejection grade. In this review, we found that the rejection episodes and rejection grade are usually related to the ischemia time, both in clinical and experimental aspects. Moreover, we summarized the potential therapeutic measures to mitigate the ischemia-reperfusion injury. Compare to static preservation, machine perfusion is a promising method that can keep VCA tissue viability and extend preservation time, which is especially beneficial for the expansion of the donor pool and better MHC-matching.

234.6: Pancreas Graft Rejection: Incidence, Treatment and Outcomes

Introduction: Pancreas rejection continues to be a major cause of graft failure (GF). Treatment protocols differ among transplant centers. Materials and Methods: Since December 2011 pancreas biopsies are performed systematically at any graft dysfunction and pancreas rejection is treated according to rejection type and grade. The objective is to report incidence, type, grade, treatment and outcomes of pancreas rejection until March, 2022. Standard anti-rejection treatment: Indeterminate and Banff 1: Steroid pulses, Banff 2: SP+ Thymoblobulin. AMR: Steroid pulses, plasmapheresis and IVIG, Mixed rejection: SP+Thymo+PP+IVIG. However, due to kidney discordant grade or rejection and/or patient condition, treatment may have varied. Results: We have performed 419 pancreas transplants at our center, of which 242 were performed during the study period. The incidence of rejection was 17% at 1y and 25% at 5y. We analyzed 93 pancreas biopsy proven rejections. Among the 71 first episodes, there were 4 indeterminate rejections (0% GF), 43 Banff 1 rejections (7% GF), 15 Banff 2 rejections (33% GF) 8 AMR (25% GF) and 1 chronic rejection (100% GF). Recurence of refection after a first episode was 11% for Banff 1 and 50% for Banff 2 and AMR cases. Among the 22 episodes of recurrent rejection there were 1 indeterminate rejection (0% GF), 14 Banff 1 rejection (7% GF), 4 Banff 2 (0 % GF), 2 AMR (100% GF) and 1 chronic rejection (100% GF). In 29 cases, a repeat biopsy was performed during the first 2 months after initial treatment confirming persistent rejection (12), resolution of rejection (16 cases) or graft failure (1 case). Discussion: Pancreas rejection is still a prevalent complication after transplantation. Tailoring anti-rejection treatment according to pancreas pathology, kidney pathology and patient status may result in a more efficient use of immunosuppression. Repeat biopsy can assist differentiate cases or persistent rejection vs resolution of rejection when elevated enzymes or hyperglycemia persists after treatment.

P8.052: Long-term Outcomes and Predictors of Graft Function in Living-Donor Preemptive Kidney Transplants

Background: Beyond the new strategies for dialysis treatment, kidney transplantation has already been associated with better outcomes for patients with advanced kidney chronic disease (CKD), and a preemptive kidney transplant seems to be the preferred option for those patients. Thus, this study purposed to evaluate predictors of long-term graft function and unfavorable outcomes in living-donor preemptive kidney transplant recipients. Methods: Single-center cohort study enrolled 222 living-donor preemptive kidney transplant recipients transplanted between 2011 and 2016 and followed up to 2021. Five-year graft function was estimated by CKD-Epi (5-year-eGFR). The primary outcome was composed of death, graft loss, acute rejection, and 5-year-eGFR < 30 mL/min/1.73m2. The multivariable analysis for 5-year-eGFR was performed by linear regression and logistic regression for the primary outcome. Results: Recipients were 40 (31; 48) years old, 59.9% were male, and 35.1% had CKD due to chronic glomerulonephritis. Donors were 48.5 (41.0; 55.2) years old, being most frequently siblings (50%) or parents (28/8%). One-third of patients received ATG as an induction strategy (30.2%), followed by Tacrolimus + azathioprine (59.5%) or cyclosporin + azathioprine (14.4%) or tacrolimus + mycophenolate (11.3%) as the maintenance immunosuppression. The overall incidence of acute rejection, graft loss, and death was 20.3%, 6.3%, and 0.5%, respectively. The median follow-up time was 88.9 months, and the median 5-year-eGFR was 54.7 mL/min. Sixty-five patients presented the primary outcome (29.2%), significantly associated with the HLA compatibility, not using induction therapy and CMV-related event. Compared with identical HLA matches, the OR was 6.81 for haploidentical (P=0.001) and 11.2 for distinct (P<0.001) HLA matches. In addition, the probability for the primary outcome was reduced by 81% (OR= 0.19, p<0.001) for patients who received ATG as induction therapy and 4-fold higher in those who had CMV-related events (OR=4.09; p=0.001). In the linear regression, the association with 5-yr-eGFR was less evident with HLA matches and AR than donor age. Thus, the identical HLA (B= +6.04; p=0.09) match and AR (B=-6.34; p=0.07) tend to be associated with 5-year-eGFR in the linear regression, while donor age (B= -0,69 per year old; p<0.001) presented a significant and inverse association. Conclusion: In recipients of living-donor preemptive kidney transplants, the HLA matches, thymoglobulin induction therapy, and CMV-related events were predictors of composite outcomes of long-term death, graft loss, acute rejection, and low grade of graft function. In addition, only donor age was independently associated with long-term graft function.

427.1: Donor Dendritic Cell Depletion Allows Novel Insights Into the Alloimmune Response Following Murine Composite Tissue and Skin Transplantation

Background: Cell-based therapies in vascularized composite tissue allotransplantation (VCA) have demonstrated promising results with the potential to modify life-long conventional immunosuppression. Dendritic cells (DCs) promote pro-inflammatory, alloimmune responses. Of particular interest is the role of DCs as antigen-presenting cells and potential tolerogenic effects as immature DCs. Material and Methods: Diphteria Toxin Receptor (DTR) transgenic or wild-type (WT) donor mice were used to deplete subsets of DCs or APCs respectively in a fully histoincompatible murine hind limb and skin transplant models; prior to procurement, all donors were either treated with diphteria toxin (DT; at -15 hours) or with clodronate (CL; 8 days prior to Tx). DBA/2J mice served as recipients. Study endpoint was on POD 6. Alloimmune response was assessed sequentially; graft survival, intragraft structural and inflammatory changes were tested serially. Results: The total number of dendritic cells (CD11b- CD11c+ DC) had markedly increased in recipients of skin WT grafts compared to recipients of VCA WT grafts (p<0.0001). The treatment with CL or DT significantly reduced the numbers of maturated and activated DC in the spleen, blood, and LN of mice receiving a skin graft, whereas numbers of CD11b-CD11c+MHCII+ and CD11b-CD11c+CD40+ DC were exclusively reduced in the spleen of VCA recipients after donor pretreatment with DT. cDC 1 subtypes were significantly higher in VCA WT vs. skin WT recipients (p<0.001). Donor pretreatment with DT decreased cDC1 counts only in recipients of VCA grafts (p<0.001).Luminex analysis revealed significantly higher TNF-alpha serum levels in the VCA WT vs. skin WT group (p<0.01). TNF-beta serum levels were significantly decreased in VCA depletion groups vs VCA WT group (p<0.001). Clinical signs of acute rejection among the VCA groups revealed higher rejection grades in the WT compared to the DC depletion group according to the BANFF criteria (grade III vs. grade II). Additional injection of immature DCs did not show any difference in allograft survival (p=ns).Conclusion: This is to our knowledge the first systematic study delineating the role of mature and immature DCs tested across and skin transplants. Those data may help explaining split-tolerance phenomenon in VCA while providing a basic concept for the utilization of mature and immature DCs in modifying alloimmune responses in VCA transplants.

Donor and Recipient Adipose-Derived Mesenchymal Stem Cell Therapy for Rat Lung Transplantation

Mesenchymal stem cells (MSCs) are beginning to be proven as immunosuppressant in the field of organ transplantation. However, the effects of MSC origin (donor or recipient) on immunosuppression are not clear. Hence, we investigated the effects of recipient and donor adipose-derived MSCs (ADMSCs) on immunosuppression in a rat lung transplantation model. Subjects were divided into no treatment, tacrolimus administration, recipient ADMSC administration, donor ADMSC administration, and mixed donor and recipient ADMSC administration groups. ADMSC-administered groups were also treated with tacrolimus. Histologic study, immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assay, and polymerase chain reaction were used for various analyses. Fluorescently labeled ADMSCs were predominant in the grafted donor lung, but not in the recipient lung, on day 5. On day 7, the pathologic rejection grades of the grafted donor lung were significantly lower in the ADMSC-administered groups (P < .05) and did not differ among these groups. Although serum hepatocyte growth factor and vascular endothelial growth factor levels did not differ among the groups, interleukin 10 level was slightly higher in the ADMSC-administered groups. The numbers of infiltrating regulatory T cells in the grafted lung were significantly higher in the ADMSC-administered groups (P < .05) but did not differ with cell origin. Transcriptional analysis suggested interleukin 6 suppression to be the main overlapping immunosuppressive mechanism, regardless of origin. Therefore, a donor or recipient origin may not influence the immunosuppressive efficacy of ADMSCs in our rat lung transplantation model. Collectively, the results indicate that allogenic ADMSCs, regardless of their origin, may exert similar immunosuppressive effects in clinical organ transplantation.

Cardiovascular Magnetic Resonance for Rejection Surveillance After Cardiac Transplantation.

Endomyocardial biopsy (EMB) is the gold standard method for surveillance of acute cardiac allograft rejection (ACAR) despite its invasive nature. Cardiovascular magnetic resonance (CMR)-based myocardial tissue characterization allows detection of myocarditis. The feasibility of CMR-based surveillance for ACAR-induced myocarditis in the first year after heart transplantation is currently undescribed. CMR-based multiparametric mapping was initially assessed in a prospective cross-sectional fashion to establish agreement between CMR- and EMB-based ACAR and to determine CMR cutoff values between rejection grades. A prospective randomized noninferiority pilot study was then undertaken in adult orthotopic heart transplant recipients who were randomized at 4 weeks after orthotopic heart transplantation to either CMR- or EMB-based rejection surveillance. Clinical end points were assessed at 52 weeks. Four hundred one CMR studies and 354 EMB procedures were performed in 106 participants. Forty heart transplant recipients were randomized. CMR-based multiparametric assessment was highly reproducible and reliable at detecting ACAR (area under the curve, 0.92; sensitivity, 93%; specificity, 92%; negative predictive value, 99%) with greater specificity and negative predictive value than either T1 or T2 parametric CMR mapping alone. High-grade rejection occurred in similar numbers of patients in each randomized group (CMR, n=7; EMB, n=8; P=0.74). Despite similarities in immunosuppression requirements, kidney function, and mortality between groups, the rates of hospitalization (9 of 20 [45%] versus 18 of 20 [90%]; odds ratio, 0.091; P=0.006) and infection (7 of 20 [35%] versus 14 of 20 [70%]; odds ratio, 0.192; P=0,019) were lower in the CMR group. On 15 occasions (6%), patients who were randomized to the CMR arm underwent EMB for clarification or logistic reasons, representing a 94% reduction in the requirement for EMB-based surveillance. A noninvasive CMR-based surveillance strategy for ACAR in the first year after orthotopic heart transplantation is feasible compared with EMB-based surveillance. HREC/13/SVH/66 and HREC/17/SVH/80. ACTRN12618000672257.

Therapeutic Drug Monitoring of Tacrolimus-Personalized Therapy in Heart Transplantation: New Strategies and Preliminary Results in Endomyocardial Biopsies

Tacrolimus (TAC) is an immunosuppressant drug approved both in the US and in the EU, widely used for the prophylaxis of organ rejection after transplantation. This is a critical dose drug: low levels in whole blood can lead to low exposure and a high risk of acute rejection, whereas overexposure puts patients at risk for toxicity and infection. Both situations can occur at whole-blood concentrations considered to be within the narrow TAC therapeutic range. We assumed a poor correlation between TAC trough concentrations in whole blood and the incidence of acute rejection; therefore, we propose to study TAC concentrations in endomyocardial biopsies (EMBs). We analyzed 70 EMBs from 18 transplant recipients at five scheduled follow-up visits during the first year post-transplant when closer TAC monitoring is mandatory. We observed five episodes of acute rejection (grade 2R) in three patients (2 episodes at 0.5 months, 2 at 3 months, and 1 at 12 months), when TAC concentrations in EMBs were low (63; 62; 59; 31; 44 pg/mg, respectively), whereas concentrations in whole blood were correct. Our results are preliminary and further studies are needed to confirm the importance of this new strategy to prevent acute rejection episodes.

Retrospective Evaluation of Rabbit Antithymocyte Globulin Induction in Heart Transplant Patients.

Background.The dosing intensity of antithymocyte globulin as induction therapy in heart transplantation remains controversial. We sought to evaluate the efficacy and safety of rabbit antithymocyte globulin at a total dose of 4.5 mg/kg compared with <4.5 mg/kg.Methods.This was a retrospective study of consecutive patients who underwent heart transplantation from January 2016 to December 2018 at a single quaternary care center. Exposure was defined as full antithymocyte globulin (4.5 mg/kg total) induction compared with partial (<4.5 mg/kg) induction. The primary outcome was the incidence of The International Society for Heart and Lung Transplantation 1990 acute cellular rejection grade 2 or above at 2 y. Secondary outcomes were all-cause mortality, number of infections, and time to therapeutic tacrolimus levels. Cox proportional hazard models were used to compare rejection rates and mortality.Results.Of 201 patients, 61 received partial and 140 received full induction. There was no difference in the cumulative incidence of cellular rejection grade 2 or above (18% versus 11.4%, P = 0.209) within 2 y. The adjusted hazard ratio was 1.45 (confidence interval: 0.62-3.37, P = 0.388) for partial compared with full induction for any grade rejection. Landmark survival analysis conditional on survival to 1 mo showed no difference in mortality (P = 0.239). There was no difference in the incidence of infection within 3 mo of transplant (partial 29.5% versus full 20.0%, P = 0.140). Both groups achieved therapeutic tacrolimus levels by day 7 after initiation.Conclusions.There was no difference in overall risk for any grade cellular rejection between partial or full dose induction therapy. Additionally, there was no difference in medium-term mortality from landmark survival analysis.

A 39-year-old male who received living kidney from his 62-year-old father, blood group matched and CDC cross match all negative, having acute cell mediated rejection 5 hours after transplant due to low Tacrolimus Trough level: A case report

A young renal transplant recipient received living kidney from his father; the Centers for Disease Control and Prevention (CDC) cross match was all negative. He had oliguria 5 hours after transplant with fever and neutrophil leukocytosis. Doppler ultrasonogram revealed increased cortical echo with increased Resistive Index suggesting acute rejection or acute tubular necrosis. Renal transplant biopsy revealed acute T cell mediated rejection (acute TCMR) Grade IIA. Trough level of Tacrolimus blood level done on Day 5 was very low; therefore, Tacrolimus dose was increased. Therapeutic level achieved after giving 6 mg twice a day with addition of erythromycin. Escalating antibiotics, increasing steroids and Tacrolimus dose saved the transplant kidney.

Validation of donor fraction cell-free DNA with biopsy-proven cardiac allograft rejection in children and adults

ObjectivesDonor-specific cell-free DNA shows promise as a noninvasive marker for allograft rejection, but as yet has not been validated in both adult and pediatric recipients. The study objective was to validate donor fraction cell-free DNA as a noninvasive test to assess for risk of acute cellular rejection and antibody-mediated rejection after heart transplantation in pediatric and adult recipients. MethodsPediatric and adult heart transplant recipients were enrolled from 7 participating sites and followed for 12 months or more with plasma samples collected immediately before all endomyocardial biopsies. Donor fraction cell-free DNA was extracted, and quantitative genotyping was performed. Blinded donor fraction cell-free DNA and clinical data were analyzed and compared with a previously determined threshold of 0.14%. Sensitivity, specificity, negative predictive value, positive predictive value, and receiver operating characteristic curves were calculated. ResultsA total of 987 samples from 144 subjects were collected. After applying predefined clinical and technical exclusions, 745 samples from 130 subjects produced 54 rejection samples associated with the composite outcome of acute cellular rejection grade 2R or greater and pathologic antibody-mediated rejection 2 or greater and 323 healthy samples. For all participants, donor fraction cell-free DNA at a threshold of 0.14% had a sensitivity of 67%, a specificity of 79%, a positive predictive value of 34%, and a negative predictive value of 94% with an area under the curve of 0.78 for detecting rejection. When analyzed independently, these results held true for both pediatric and adult cohorts at the same threshold of 0.14% (negative predictive value 92% and 95%, respectively). ConclusionsDonor fraction cell-free DNA at a threshold of 0.14% can be used to assess for risk of rejection after heart transplantation in both pediatric and adult patients with excellent negative predictive value.

Cardiac Transplantation Italian Allocation System Analysis: Single Center Results

<h3>Purpose</h3> Analyze the characteristic of donors and recipients in our center and the results of heart transplantations for the two different levels of priority according to Italian previous allocation system, urgent patients and elective ones. <h3>Methods</h3> From 2006 to August 2020, 306 heart transplantations were performed at our institution. Of them 54 (17.6%) in urgency. Reasons for urgency were: 24 patients on ECMO support, 8 on IABP and 13 on intravenous inotropes, 4 arrhythmic storms and 5 complicated LVAD. <h3>Results</h3> In the urgent population, recipients were predominantly male (68.5 % vs 77.4 % in the elective p=0.17) and significantly younger (46.4 vs 52.2 years, p=0.001). More patients were on ECMO support (42.6% vs 1.7% p=0.000), IABP (42.6 vs 6.7% p=0.000) and mechanically ventilated (46.2% vs 6% p=0.000) pre transplant. The donors of urgent patients were younger than donors of the elective ones (38.9 vs 42.4 years (p=0.06). Donor's gender (68% males) and cause of brain death (brain trauma 44,4%, and cerebral hemorrhage 35,2%) were comparable in the two groups. Mean ischemic time was longer in urgent cases (211 vs 191 minutes, p=0.01) as donations came from across the whole country. Severe Early Graft Failure (EGF) requiring ECMO occurred in 9.4% of patients in the urgent population vs 3.6% in the elective with no significant difference (p=0.076), as well as in hospital mortality that was 11.5% in urgent vs 8.3% (p=0.429). Recipients' risk factors for in-hospital mortality were age [OR 1.05 (1.01;1.09), p=0.028] and Glomerular Filtration Rate (GFR) < 40 ml/min [2.68 (1.04;6.92), p=0.047]. No donors' variable resulted to be a risk factor for in-hospital mortality. Episodes and grade of rejection did not diverge between groups, and so did follow up mortality (16.7% in urgent vs 22.4% in electives p=0.444) Mean follow up time for urgent population was shorter (53.6 vs 75.6 months (p=0.007). Overall 5 and 10 years' mortality was 79.7% and 62% vs 88.1% and 72.8% for urgent and elective patients respectively (p=0.721). <h3>Conclusion</h3> This retrospective analysis of our transplantation activity with the different priority regimen shows little differences in terms of mortality and complications. Despite the worse clinical characteristics of the urgent recipients and the longer ischemic time, results do not significantly differ from the elective population, underlying the importance of donation quality.

Real World Use of Extracorporeal Photopheresis After Heart Transplantation - Clinical Outcomes from a Seven Centre European Study

<h3>Purpose</h3> Extracorporeal photopheresis (ECP) is recommended by different apheresis and transplant societies based on study results from 1998 and 2006 as an adjunctive therapy in prevention and treatment of acute cellular rejection (ACR) after heart transplantation (HTx). At present, however, it is also used to treat antibody mediated rejection (AMR) with and without (+/-) donor specific antibodies (DSA). The aim of this study was to describe the real-world use of ECP across European heart transplant centres and assess its impact on clinical outcomes in the modern era of heart transplantation. <h3>Methods</h3> Seven transplant centres located in five European countries participated in this retrospective, explorative, single-arm chart review study of over 90 patients. All included patients received ECP after heart transplant in 2015 or later with a follow-up period of up to two years after the last ECP treatment. Data have been extracted from the medical charts covering patient characteristics, reasons for ECP treatment, ECP treatment schedule and duration, concomitant treatments, clinical outcomes (e.g. graft dysfunctions, rejections, survival, immune system, kidney function) as well as treatment related complications and safety. <h3>Endpoints</h3> The findings of the full study analysis from the largest known multi-centre study on ECP after HTx, will be presented for ECP treatment overall, and for the three sub-groups ACR, AMR (+/- DSA), and prevention of rejection. Endpoints that will be reported will include graft and overall survival, incidence of rejections, infections and complications, changes in rejection grading, improvements in graft function, and ECP related safety data.

Acute 2R Cellular Rejection on Routine Surveillance Endomyocardial Biopsy is Not Associated with Significant Hemodynamic Changes on Right Heart Catheterization

<h3>Purpose</h3> Routine surveillance endomyocardial biopsy (EMB) following heart transplantation (HT) is considered an important tool for early detection of allograft rejection. However, the recommended frequency of EMB is not defined by societal guidelines and scheduling practices are widely variable between transplant centers. We sought to identify whether there were differences in hemodynamic, echocardiographic or outcome parameters between patients categorized by rejection grade on surveillance EMB. <h3>Methods</h3> We examined LVEF by echocardiography and right heart catheterization (RHC) hemodynamic parameters obtained at the time of routine surveillance EMB in 222 patients who underwent HT between January 2015 - January 2021 at a single center. <h3>Results</h3> On the first four post-transplant paired EMBs and RHCs, similar hemodynamic measurements and LVEFs were observed regardless of corresponding cellular rejection grade (P > 0.5) (Table 1). Patients were then grouped by if they ever had a biopsy with significant rejection (SR) defined as ≥ grade 2R (n=119, 54%) or no significant rejection (NSR) defined as grade 0 or 1R (n=103, 46%). Kaplan-Meier survival analysis showed no significant difference in post-transplant survival (HR 0.56, p=0.14) in SR vs NSR (Figure 1). A total of 26 deaths were observed (16 NSR and 10 SR). For those that died in the SR group, only one may have been related to allograft rejection with the cause of death being outside hospital sudden death. In a SR sub-group of 39 patients with ≥3 episodes of grade 2R mortality was low (n=3, 8%). <h3>Conclusion</h3> We observed minimal hemodynamic, echocardiographic or prognostic implications of cellular rejection detected by routine surveillance EMB. These findings lend support to the practice of limiting high frequency routine surveillance EMB in post-HT patients, even in the early period when non-invasive diagnostics are not applicable.

Prognostic Implications and Characteristics of Low dd-cfDNA Results in Heart Transplant Patients with Biopsy Proven Rejection

<h3>Purpose</h3> Donor-derived cell-free DNA (dd-cfDNA) has emerged as an important noninvasive marker of heart transplant allograft injury. The characteristics and prognostic implications of low level dd-cfDNA in patients with histological evidence of acute rejection is not known. Although considered the gold standard, endomyocardial biopsy is limited to focal tissue sampling and is subject to interobserver variability in its interpretation. <h3>Methods</h3> Patients enrolled in the Surveillance HeartCare Outcomes Registry (SHORE) with biopsy proven acute cellular rejection (ACR) and antibody mediated rejection (AMR) who had low levels of dd-cfDNA (<0.15%) were evaluated. A targeted amplification, next generation-sequencing assay (AlloSure®; CareDx, Inc.) was used to detect dd-cfDNA. <h3>Results</h3> The SHORE registry had 2029 patients in total. Patients with ACR 1R were excluded. Only those patients who had a dd-cfDNA level within 30 days of biopsy (mean 2.7 days) were included. 61 patients (male 78%, median age 48 years) had low dd-cfDNA and endomyocardial biopsy evidence of ACR ≥2R and/or AMR ≥1R. Mean duration of the biopsy from the time of transplant was 196.31 days (IQR 88-257 days). ACR was seen in 36 patients (35=2R, 1=3R) and AMR was seen in 27 patients (21=pAMR 1(H+), 6=pAMR2). 2 patients had both ACR and AMR. The mean ejection fraction (EF) at the time of the rejection was mostly preserved (ACR 2R=EF 62.8±5.6%,ACR 3R=EF 47.5%, and AMR 1=EF 60.9±6%, AMR 2=EF 61±4.7%). 2 patients (3.3%) died 330±31days after the initial diagnosis of rejection. None of the patients developed graft dysfunction at one year follow up. One patient developed coronary artery vasculopathy and 7 patients developed <i>de novo</i> DSA. <h3>Conclusion</h3> Patients with low dd-cfDNA and biopsy-confirmed rejection typically had lower grades of rejection and maintained preserved left ventricular function on echocardiography. There was no immediate death after rejection or graft dysfunction. In this preliminary analysis, low dd-cfDNA despite biopsy evidence of rejection appears to have favorable prognosis. Discordance between light-based histology and molecular histology has shown better correlation with dd-cfDNA levels, and so consideration of dd-cfDNA in treatment and immunosuppression management requires ongoing evaluation.

Validation of Systematic Histologic Reporting of Transbronchial Biopsy Eosinophils for Sensitivity of Detection and Associations with Early Outcomes After Lung Transplant

<h3>Purpose</h3> Eosinophils are reported in 8% of transbronchial biopsies (TBBx) and their presence is associated with an increased risk of CLAD and death. We aimed to validate a systematic reporting scheme for the presence, gradation, and pattern of TBBx eosinophils. <h3>Methods</h3> A prospective reporting scheme for TBBx eosinophils was introduced at St Vincent's Sydney in Jan-20. All TBBx were reported by blinded pathologists for the presence of eosinophils, histologic pattern (peribronchial, interstitial, perivascular) and count/high power field (HPF). Standardised reporting was compared with a historic TBBx cohort from Jan-Dec 2019. TBBx were classified for cellular rejection as per ISHLT guidelines and positive bronchoalveolar lavage (BAL) microbiology/eosinophils and blood eosinophil count was recorded. Multivariable logistic regression measured associations with an increased risk of TBBx eosinophils. <h3>Results</h3> A total of 213 TBBx from 93 lung transplant recipients were systematically reviewed, with 61.5% as surveillance procedures. The mean (SD) recipient age was 52 (13), with 96 (45%) males, at median (IQR) 85 (475) days after transplant. TBBx eosinophils were detected in greater proportions with systematic (27.7%) compared to historic reporting (5.6%) p<0.001. When present, eosinophils were detected in median (IQR) 18.0% (19) biopsy specimens with median (IQR) 2 (5) cell/HPF. TBBx eosinophil patterns were reported in the following proportions; Interstitial 64%, perivascular 7%, peribronchial 25%. In univariable analysis, A grade (OR 3.11 p<0.01), blood eosinophils (OR 13.28 p=0.04) and BAL microbiology (OR 2.15 p=0.02) were associated with increased risk of TBBx eosinophils, however BAL eosinophils (OR 1.75 p=0.31) were not. In multivariable analysis, A grade (OR 2.93 95%CI 1.65-5.20) and positive BAL microbiology (OR 2.18 95%CI 1.10-4.31) were associated with an increased risk of TBBx eosinophils. <h3>Conclusion</h3> Systematic reporting of TBBx eosinophils improves sensitivity for detection compared with historic reporting. Acute vascular rejection grades and positive BAL microbiology were independently associated with an increased risk of TBBx eosinophils after adjustment for blood eosinophil count. Systematic reporting of TBBx eosinophils may identify patients at risk of poor outcomes.

Fibrosis of Chronic Lung Allograft Dysfunction After Orthotopic Lung Transplantation is Prevented in the Absence of Secondary Lymphoid Organs in a Mouse Model

<h3>Purpose</h3> It was reported that acute lung allograft rejection occurred without secondary lymphoid organs (SLOs) in a mouse allogenic lung transplantation model. The objective of this study was to investigate the role of SLOs in chronic lung allograft dysfunction (CLAD) after allogenic mouse lung transplantation using splenectomized alymphoplastic (aly/aly) mice as SLOs-deficient recipient. <h3>Methods</h3> Left lung of BALB/c mouse was transplanted into C57BL/6J (B6) or aly/aly mouse with B6 background. Low-dose cyclosporine (10 mg/kg, 3 times/week) and methylprednisolone (1.6 mg/kg, 3 times/week) were given in all the recipient mice until sacrifice. The recipient mice were sacrificed at day 15 (D15, n=4, each group) or day 30 (D30, n=5, each group) to evaluate histology. <h3>Results</h3> Allograft in B6 mice showed A2-3 rejection on D15 and A2-3 rejection with severe peri-airway and peri-vascular fibrosis on D30. On the contrary, allograft in aly/aly mouse showed A1-2 rejection on D15 and D30 without fibrosis (Figure 1). Quantification of peri-airway fibrosis thickness demonstrated significantly less fibrotic change in aly/aly mice than B6 mice on D30 (p = 0.012) (Figure 2). The A grade rejection score was significantly low on D30 in aly/aly mice (median A1, range A1-A2) than B6 mice (median A3, range A2-A3) (p = 0.015). <h3>Conclusion</h3> Fibrotic change with cellular rejection did not occur in the absence of SLOs in a mouse model of CLAD induced by inadequate immunosuppression, which may provide a platform for novel strategy to prevent CLAD.

Utility of AlloMap® Scores in Younger Pediatric Heart Transplant Recipients

<h3>Purpose</h3> AlloMap® is used to predict acute cellular rejection (ACR) post heart transplant (HT) in pediatric patients > 15 years old. Data on its utility in a younger pediatric cohort are limited. We examine the correlation of AlloMap® score with hemodynamic, echocardiographic (echo), and endomyocardial biopsy (EMB) markers of rejection in HT recipients ≤ 15 years old. <h3>Methods</h3> Retrospective study of all pediatric patients ≤ 15 years old at our institution who had AlloMap® testing with routine cardiac catheterization and EMB, or when rejection was suspected based on clinical/echo evidence. <h3>Results</h3> 438 AlloMap® scores obtained from 57 children from 2010-2017. Median age at HT was 2.4 years (IQR 0.6-11.9 years) and 56% (n=32) were male. Indication for HT was cardiomyopathy (CM) in 56.1% (n=32) and congenital heart disease (CHD) in 43.9% (n=25) of patients. AlloMap® scores did not differ between CHD and CM groups (32.8 ± 3.5 vs 32.4 ± 3.7, p=.2). Scores were highest in those <2 years old. Scores differed between patients aged < 2 versus 2-10 years (p<.001) and between patients aged 2-10 and > 10 years old (p<.001). 32 rejection episodes were identified in 18 patients; 5 patients (27.8%) were < 1 year old. ACR grade 0/1R and 2R/3R were seen in 84.4% (n=27) and 15.6% (n=5), respectively. Those with ACR 0R had lower AlloMap® scores than those with ACR 1R (p=.024), but there was no difference between 1R and higher grade rejection. AlloMap® score weakly correlated with left ventricular end diastolic pressure (LVEDP) (r=.149, p=.026), but not with echo variables. <h3>Conclusion</h3> AlloMap® scores are similar in CM and CHD patients. Scores decrease with age, which may represent differing gene expression profiles over time. Scores are lower for those with ACR 0R versus 1R, but do not differ significantly between 1R and higher grade ACR. AlloMap® score correlates weakly with LVEDP, but not echo variables. Sample size is small with few rejection episodes. A larger study is needed to determine the utility of routine AlloMap® surveillance in HT recipients ≤ 15 years of age.

Impact of Liver Biopsy Size on Histopathologic Evaluation of Liver Allograft Rejection.

Allograft liver biopsy is the gold standard in assessing transplant recipients for graft dysfunction. The impact of biopsy sample size on the diagnosis of acute cellular rejection (ACR) has not been studied. To assess the relationship of biopsy sample length with the diagnosis and determine optimal biopsy sample size in the transplant setting. We retrospectively reviewed 68 core biopsies from patients with a history of liver transplant. Each biopsy sample was read, on 5 different occasions with differing lengths, to assess for ACR per Banff criteria. Categorical agreement was calculated from rejection severity. The length of biopsy sample strongly correlated with the number of portal tracts. ACR rates increased from 73.5% to 79.4% with increase in length from 1 cm to 2 cm, and moderate rejection increased from 27.9% to 33.82%. At 1.0 and 1.5 cm, no cases of severe rejection were detected; at 2.0 cm, 1 case was detected; and at 3.0 cm, 2 cases were detected. The major error rate was reduced to less than 10% with a length of 2.0 cm, at which length the average number of complete and partial portal triads was 10 and 13, respectively. The likelihood of diagnosing ACR and rejection grade increased substantially with increase in biopsy sample length. This study suggests that a minimum length of 2 cm, 10 complete portal triads, or 13 partial/complete portal triads should be obtained for confident exclusion and grading of ACR.